Xiaotan Sanjie decoction attenuates tumor angiogenesis by manipulating Notch-1-regulated proliferation of gastric cancer stem-like cells

AIM: To determine the underlying mechanisms of action and influence of Xiaotan Sanjie (XTSJ) decoction on gastric cancer stem-like cells (GCSCs).METHODS: The gastric cancer cell line MKN-45 line was selected and sorted by FACS using the cancer stem cell marker CD44; the stemness of these cells was checked in our previous study. In an in vitro study, the expression of Notch-1, Hes1, Vascular endothelial growth factor (VEGF), and Ki-67 in both CD44-positive gastric cancer stem-like cells (GCSCs) and CD44-negative cells was measured by Western blot. The effect of XTSJ serum on cell viability and on the above markers was measured by MTT assay and Western blot, respectively. In an in vivo study, the ability to induce angiogenesis and maintenance of GCSCs in CD44-positive-MKN-45- and CD44-negative-engrafted mice were detected by immunohistochemical staining using markers for CD34 and CD44, respectively. The role of XTSJ decoction in regulating the expression of Notch-1, Hes1, VEGF and Ki-67 was measured by Western blot and real-time polymerase chain reaction.RESULTS: CD44⁺ GCSCs showed more cell proliferation and VEGF secretion than CD44-negative cells in vitro, which were accompanied by the high expression of Notch-1 and Hes1 and positively associated with tumor growth (GCSCs vs CD44-negative cells, 2.72 ± 0.25 vs 1.46 ± 0.16, P < 0.05) and microvessel density (MVD) (GCSCs vs CD44-negative cells, 8.15 ± 0.42 vs 3.83 ± 0.49, P < 0.001) in vivo. XTSJ decoction inhibited the viability of both cell types in a dose-dependent manner in vitro. Specifically, a significant difference in the medium- (82.87% ± 6.53%) and high-dose XTSJ groups (77.43% ± 7.34%) was detected at 24 h in the CD44⁺ GCSCs group compared with the saline group (95.42% ± 5.76%) and the low-dose XTSJ group (90.74% ± 6.57%) (P < 0.05). However, the efficacy of XTSJ decoction was reduced in the CD44^- groups; significant differences were only detected in the high-dose XTSJ group at 48 h (78.57% ± 6.94%) and 72 h (72.12% ± 7.68%) when compared with the other CD44- groups (P < 0.05). Notably, these differences were highly consistent with the Notch-1, Hes1, VEGF and Ki-67 expression in these cells. Similarly, in vivo, XTSJ decoction inhibited tumor growth in a dose-dependent manner. A significant difference was observed in the medium- (1.76 ± 0.15) and high-dose XTSJ (1.33 ± 0.081) groups compared with the GCSCs control group (2.72 ± 0.25) and the low-dose XTSJ group (2.51 ± 0.25) (P < 0.05). We also detected a remarkable decrease of MVD in the medium- (7.10 ± 0.60) and high-dose XTSJ (5.99 ± 0.47) groups compared with the GCSC control group (8.15 ± 0.42) and the low-dose XTSJ group (8.14 ± 0.46) (P < 0.05). Additionally, CD44 expression was decreased in these groups [medium- (4.43 ± 0.45) and high-dose XTSJ groups (3.56 ± 0.31) vs the GCSC control (5.96 ± 0.46) and low dose XTSJ groups (5.91 ± 0.38)] (P < 0.05). The significant differences in Notch-1, Hes1, VEGF and Ki-67 expression highly mirrored the results of XTSJ decoction in inhibiting tumor growth, MVD and CD44 expression.CONCLUSION: Notch-1 may play an important role in regulating the proliferation of GCSCs; XTSJ decoction could attenuate tumor angiogenesis, at least partially, by inhibiting Notch-1.     

Perioperative restricted fluid therapy preserves immunological function in patients with colorectal cancer

AIM: To investigate the effect of perioperative restricted fluid therapy on circulating CD4⁺/CD8⁺ T lymphocyte ratio, percentage of regulatory T cells (Treg) and postoperative complications in patients with colorectal cancer.METHODS: A total of 185 patients met the inclusion criteria and were included in the randomized clinical trial. These patients were divided into two groups according to receipt of either perioperative standard (S, n = 89) or restricted (R, n = 96) fluid therapy. Clinical data of these patients were collected in this prospective study. Perioperative complications and cellular immunity changes (CD4⁺/CD8⁺ and Treg) were analyzed comparatively between the two groups.RESULTS: Both during surgery and on postoperative days, the total volumes of fluids administered in the R group were significantly lower than those in the S group (1620 ± 430 mL vs 3110 ± 840 mL; 2090 ± 360 mL vs 2750 ± 570 mL; 1750 ± 260 mL vs 2740 ± 490 mL; 1620 ± 310 mL vs 2520 ± 300 mL; P < 0.05). Decreased ratios of circulating CD4⁺/CD8⁺ T lymphocytes (1.47 ± 0.28 vs 2.13 ± 0.26; 1.39 ± 0.32 vs 2.21 ± 0.24; P < 0.05) and Treg percentage values (2.79 ± 1.24 vs 4.26 ± 1.04; 2.46 ± 0.98 vs 4.30 ± 1.12; P < 0.05) were observed after surgery in both groups. However, in the R group, these values restored more quickly starting from postoperative day 2 (1.44 ± 0.24 vs 1.34 ± 0.27; 2.93 ± 1.08 vs 2.52 ± 0.96; P < 0.05). The proportion of patients with complications was significantly lower in the restricted group (36 of 89 vs 59 of 96, P < 0.01).CONCLUSION: Perioperative restricted intravenous fluid regimen leads to a low postoperative complication rate and better cellular immunity preservation in patients with colorectal cancer.     

Increased density of tolerogenic dendritic cells in the small bowel mucosa of celiac patients

AIM: To investigate the densities of dendritic cells(DCs) and FOXP3+ regulatory T cells(Tregs) and their interrelations in the small bowel mucosa in untreated celiac disease(CD) patients with and without type 1 diabetes(T1D).METHODS: Seventy-four patients(45 female, 29 male, mean age 11.1 ± 6.8 years) who underwent small bowel biopsy were studied. CD without T1 D was diagnosed in 18 patients, and CD with T1 D was diagnosed in 15 patients. Normal small bowel mucosa was found in two T1 D patients. Thirty-nine patients(mean age 12.8 ± 4.9 years) with other diagnoses(functional dyspepsia, duodenal ulcer, erosive gastritis, etc.) formed the control group. All CD patients had partial or subtotal villous atrophy according to the Marsh classification: Marsh grade Ⅲa in 9, grade Ⅲb in 21 and grade Ⅲc in 3 cases. Thirty-nine patients without CD and 2 with T1 D had normal small bowel mucosa(Marsh grade 0). The densities of CD11c+, IDO+, CD103+, Langerin(CD207+) DCs and FOXP3+ Tregs were investigated by immunohistochemistry(on paraffin-embedded specimens) and immunofluorescence(on cryostat sections) methods using a combination of mono- and double-staining. Sixtysixserum samples were tested for Ig A-tissue transglutaminase(t TG) using a fully automated Eli ATM Celikey Ig A assay(Pharmacia Diagnostics, Freiburg, Germany). RESULTS: The density of CD11c+ DCs was significantly increased in CD patients compared with patients with normal mucosa(21.67 ± 2.49 vs 13.58 ± 1.51, P = 0.007). The numbers of FOXP3+ cells were significantly higher in CD patients(10.66 ± 1.50 vs 1.92 ± 0.37, P = 0.0002) and in patients with CD and coexisting T1D(8.11 ± 1.64 vs 1.92 ± 0.37, P = 0.002) compared with patients with normal mucosa. The density of FOXP3+ cells significantly correlated with the histologicalgrade of atrophic changes in the small bowel mucosa according to the March classification(r = 0.62; P 0.0001) and with levels of Ig A antibody(r = 0.55; P 0.0001). The densities of IDO+ DCs were significantly higher in CD patients(21.6 ± 2.67 vs 6.26 ± 0.84, P = 0.00003) and in patients with CD and coexisting T1D(19.08 ± 3.61 vs 6.26 ± 0.84, P = 0.004) compared with patients with normal mucosa. A significant correlation was identified between the densities of IDO+ DCs and FOXP3+ T cells(r = 0.76; P = 0.0001). The mean values of CD103+ DCs were significantly higher in CD patients(10.66 ± 1.53 vs 6.34 ± 0.61, P = 0.01) and in patients with CD and associated T1D(11.13 ± 0.72 vs 6.34 ± 0.61, P = 0.00002) compared with subjects with normal small bowel mucosa. The mean value of Langerin+ DCs was higher in CD patients compared with persons with normal mucosa(7.4 ± 0.92 vs 5.64 ± 0.46, P = 0.04).CONCLUSION: The participation of diverse DC subsets in the pathological processes of CD and the possible involvement of tolerogenic DCs in Tregs development to maintain intestinal immunological tolerance in CD patients are revealed.     

Autologous mobilized peripheral blood CD34+ cell infusion in non-viral decompensated liver cirrhosis

AIM: To study the effect of mobilized peripheral blood autologous CD34 positive(CD34+) cell infusion in patients with non-viral decompensated cirrhosis.METHODS: Cirrhotic patients of non-viral etiology were divided into 2 groups based on their willingness to be listed for deceased donor liver transplant(DDLT)(control, n = 23) or to receive autologous CD34+ cell infusion through the hepatic artery(study group, n= 22). Patients in the study group were admitted to hospital and received granulocyte colony stimulating factor injections 520 μg/d for 3 consecutive days to mobilize CD34+ cells from the bone marrow. On day 4,leukapheresis was done and CD34+ cells were isolated using CliniMAC magnetic cell sorter. The isolated CD34+ cells were infused into the hepatic artery under radiological guidance. The patients were discharged within 48 h. The control group received standard of care treatment for liver cirrhosis and were worked up for DDLT as per protocol of the institute. Both groups were followed up every week for 4 wk and then every month for 3 mo.RESULTS: In the control and the study group, the cause of cirrhosis was cryptogenic in 18(78.2%) and16(72.72%) and alcohol related in 5(21.7%) and6(27.27%), respectively. The mean day 3 cell count(cells/μL) was 27.00 ± 20.43 with a viability of 81.84± 11.99%. and purity of 80%-90%. Primary end point analysis revealed that at 4 wk, the mean serum albumin in the study group increased significantly(2.83± 0.36 vs 2.43 ± 0.42, P = 0.001) when compared with controls. This improvement in albumin was,however, not sustained at 3 mo. However, at the end of3 mo there was a statistically significant improvement in serum creatinine in the study group(0.96 ± 0.33 vs 1.42 ± 0.70, P = 0.01) which translated into a significant improvement in the Model for End-Stage Liver Disease score(15.75 ± 5.13 vs 19.94 ± 6.68,P = 0.04). On statistical analysis of secondary end points, the transplant free survival at the end of 1 mo and 3 mo did not show any significant difference(P =0.60) when compared to the control group. There was no improvement in aspartate transaminase, alanine transaminase, and bilirubin at any point in the study population. There was no mortality benefit in the study group. The procedure was safe with no procedural or treatment related complications.CONCLUSION: Autologous CD 34+ cell infusion is safe and effectively improves liver function in the short term and may serve as a bridge to liver transplantation.     

Epstein-Barr virus is related with 5-aminosalicylic acid,tonsillectomy, and CD19+ cells in Crohn’s disease

AIM: To study anti-Epstein-Barr virus (EBV) IgG antibodies in Crohn´s disease in relation to treatment, immune cells, and prior tonsillectomy/appendectomy.METHODS: This study included 36 CD patients and 36 healthy individuals (controls), and evaluated different clinical scenarios (new patient, remission and active disease), previous mucosa-associated lymphoid tissue removal (tonsillectomy and appendectomy) and therapeutic regimens (5-aminosalicylic acid, azathioprine, anti-tumor necrosis factor, antibiotics, and corticosteroids). T and B cells subsets in peripheral blood were analyzed by flow cytometry (markers included: CD45, CD4, CD8, CD3, CD19, CD56, CD2, CD3, TCRαβ and TCRγδ) to relate with the levels of anti-EBV IgG antibodies, determined by enzyme-linked immunosorbent assay.RESULTS: The lowest anti-EBV IgG levels were observed in the group of patients that were not in a specific treatment (95.4 ± 53.9 U/mL vs 131.5 ± 46.2 U/mL, P = 0.038). The patients that were treated with 5-aminosalicylic acid showed the highest anti-EBV IgG values (144.3 U/mL vs 102.6 U/mL, P = 0.045). CD19⁺ cells had the largest decrease in the group of CD patients that received treatment (138.6 vs 223.9, P = 0.022). The analysis of anti-EBV IgG with respect to the presence or absence of tonsillectomy showed the highest values in the tonsillectomy group of CD patients (169.2 ± 20.7 U/mL vs 106.1 ± 50.3 U/mL, P = 0.002). However, in the group of healthy controls, no differences were seen between those who had been tonsillectomized and subjects who had not been operated on (134.0 ± 52.5 U/mL vs 127.7 ± 48.1 U/mL, P = 0.523).CONCLUSION: High anti-EBV IgG levels in CD are associated with 5-aminosalicylic acid treatment, tonsillectomy, and decrease of CD19⁺ cells.     

B1a lymphocytes in the rectal mucosa of ulcerative colitis patients

AIM:To assess B1a cell expression in the rectal mucosa of ulcerative colitis (UC) patients in comparison with healthy controls.METHODS:Rectal mucosa biopsies were collected from 15 UC patients and 17 healthy controls.CD5 + B cells were analysed by three colour flow cytometry from rectal mucosal samples after mechanical disaggregation by Medimachine.Immunohistochemical analysis of B and T lymphocytes was also performed.Correlations between,on the one hand,rectal B1a cell concentrations and,on the other,erythrocyte sedimentation rate and C-reactive protein levels and clinical,endoscopic and histological disease activity indices were evaluated.RESULTS:Rectal B-lymphocyte (CD19 + /CD45 +) rate and concentration were higher in UC patients compared with those in healthy controls (47.85% ± 3.12% vs 26.10% ± 3.40%,P=0.001 and 501 ± 91 cells/mm 2 vs 117 ± 18 cells/mm 2,P 0.001);Rectal B1a cell density (CD5 + CD19 +) was higher in UC patients than in healthy controls (85 ± 15 cells/mm 2 vs 31 ± 6.7 cells/mm 2,P=0.009).Rectal B1a cell (CD5/CD19 +) rate correlated inversely with endoscopic classification (Rs=-0.637,P 0.05).CONCLUSION:B1a lymphocytes seem to be involved in the pathogenesis of UC,however,the role they play in its early phases and in disease activity,have yet to be defined.     

Hepatocyte isolation from resected benign tissues: Results of a 5-year experience

AIM To analyze retrospectively a 5-year experience of human hepatocyte isolation from resected liver tissues with benign disease.METHODS We established a method of modified four-step retrograde perfusion to isolate primary human hepatocytes. Samples were collected from the resected livers of patients with intrahepatic duct calculi(n = 7) and liver hemangioma(n = 17). Only the samples weighing ≥ 15 g were considered suitable for hepatocyte isolation. By using the standard trypan blue exclusion technique, hepatocyte viability and yield were immediately determined after isolation.RESULTS Twenty-four liver specimens, weighing 15-42 g, were immediately taken from the margin of the removed samples and transferred to the laboratory for hepatocyte isolation. Warm ischemia time was 5-35 min and cold ischemia time was 15-45 min. For the 7 samples of intrahepatic duct calculi, the method resulted in a hepatocyte yield of 3.49 ± 2.31 × 10~6 hepatocytes/g liver, with 76.4% ± 10.7% viability. The 17 samples of liver hemangioma had significantly higher yield of cells(5.4 ± 1.71 × 10~6 cells/g vs 3.49 ± 2.31 × 10~6 cells/g, P 0.05) than the samples of intrahepatic duct calculi. However, there seems to be no clear difference in cell viability(80.3% ± 9.67% vs 76.4% ± 10.7%, P 0.05). We obtained a cell yield of 5.31 ± 1.87 × 10~6 hepatocytes/g liver when the samples weighed 20 g. However, for the tissues weighing ≤ 20 g, a reduction in yield was found(3.08 ± 1.86 × 10~6 cells/g vs 5.31 ± 1.87 × 10~6 cells/g, P 0.05).CONCLUSION Benign diseased livers are valuable sources for largenumber hepatocyte isolation. Our study represents the largest number of primary human hepatocytes isolated from resected specimens from patients with benign liver disease. We evaluated the effect of donor liver characteristics on cell isolation, and we found that samples of liver hemangioma can provide better results than intrahepatic duct calculi, in terms of cell yield. Furthermore, the size of the tissues can affect the outcome of hepatocyte isolation.     

Suppression of colorectal cancer metastasis by nigericin through inhibition of epithelial-mesenchymal transition

AIM: To evaluate the effect of nigericin on colorectal cancer and to explore its possible mechanism.METHODS: The human colorectal cancer (CRC) cell lines HT29 and SW480 were treated with nigericin or oxaliplatin under the conditions specified. Cell viability assay and invasion and metastasis assay were performed to evaluate the effect of nigericin on CRC cells. Sphere-forming assay and soft agar colony-forming assay were implemented to assess the action of nigericin on the cancer stem cell properties of CRC cells undergone epithelial-mesenchymal transition (EMT).RESULTS: Compared with oxaliplatin, nigericin showed more toxicity for the HT29 cell line (IC50, 12.92 ± 0.25 μmol vs 37.68 ± 0.34 μmol). A similar result was also obtained with the SW116 cell line (IC50, 15.86 ± 0.18 μmol vs 41.02 ± 0.23 μmol). A Boyden chamber assay indicated that a significant decrease in the number of HT29 cells migrating through polyvinylidene fluoride membrane was observed in the nigericin-treated group, relative to the vehicle-treated group [11 ± 2 cells per high-power field (HPF) vs 19.33 ± 1.52 cells per HPF, P < 0.05]. Compared to the control group, the numbers of HT29 cells invading through the Matrigel-coated membrane also decreased in the nigericin-treated group (6.66 ± 1.52 cells per HPF vs 14.66 ± 1.52 cells per HPF, P < 0.05). Nigericin also reduced the proportion of CD133⁺ cells from 83.57% to 63.93%, relative to the control group (P < 0.05). Nigericin decreased the number of spheres relative to the control group (0.14 ± 0.01 vs 0.35 ± 0.01, P < 0.05), while oxaliplatin increased the number of spheres relative to the control group (0.75 ± 0.02 vs 0.35 ± 0.01; P < 0.05). Nigericin also showed a decreased ability to form colonies under anchorage-independent conditions in a standard soft agar assay after 14 d in culture, relative to the control group (1.66 ± 0.57 vs 7 ± 1.15, P < 0.05), whereas the colony numbers were higher in the oxaliplatin group relative to the vehicle-treated controls (14.33 ± 0.57 vs 7 ± 1.15, P < 0.05). We further detected the expression of E-cadherin and vimentin in cells treated with nigericin and oxaliplatin. The results showed that HT29 cells treated with nigericin induced an increase in E-cadherin expression and a decrease in the vimentin expression relative to vehicle controls. In contrast, oxaliplatin downregulated the expression of E-cadherin and upregulated the expression of vimentin in HT29 cells relative to vehicle controls.CONCLUSION: This study demonstrated that nigericin could partly reverse the EMT process during cell invasion and metastasis.     

Esophageal Helicobacter pylori colonization aggravates esophageal injury caused by reflux

AIM: To investigate esophageal Helicobacter pylori (H. pylori) colonization on esophageal injury caused by reflux and the related mechanisms.METHODS: An esophagitis model, with acid and bile reflux, was surgically produced in male rats. The rats were randomly divided into either: (1) an esophagogastroduodenal anastomosis (EGDA) group; (2) an EGDA with H. pylori infection group; (3) a pseudo-operation with H. pylori infection group; or (4) a pseudo-operation group. All rats were kept for 36 wk. Based on the location of H. pylori colonization, the EGDA rats with H. pylori infection were subdivided into those with concomitant esophageal H. pylori colonization or those with only gastric H. pylori colonization. The esophageal injuries were evaluated grossly and microscopically. The expressions of CDX2 and MUC2 were determined by real-time polymerase chain reaction (RT-PCR) and immunohistochemistry. Ki-67 antigen expression was determined by immunohistochemistry. The mRNA levels of cyclin D1, c-Myc, Bax and Bcl-2 were determined by RT-PCR. Cell apoptosis was evaluated using the TdT-mediated dUTP nick-end labeling method.RESULTS: Esophagitis, Barrett’s esophagus (BE), and esophageal adenocarcinoma (EAC) developed in rats that underwent EGDA. When comparing rats with EGDA and concomitant esophageal H. pylori colonization to EGDA-only rats, the severity of injury (87.9 ± 5.2 vs 77.2 ± 8.6, macroscopically, 92.5 ± 8.0 vs 83.8 ± 5.5, microscopically, both P < 0.05) and the incidences of BE (80.0% vs 33.3%, P = 0.055) and EAC (60.0% vs 11.1%, P < 0.05) were increased. These increases were associated with upregulation of CDX2 and MUC2 mRNA (10.1 ± 5.4 vs 3.0 ± 2.9, 8.4 ± 4.6 vs 2.0 ± 3.2, respectively, Ps < 0.01) and protein (8.1 ± 2.3 vs 3.3 ± 3.1, 7.3 ± 4.0 vs 1.8 ± 2.7, respectively, all P < 0.05). The expression of Ki-67 (8.9 ± 0.7 vs 6.0 ± 1.7, P < 0.01) and the presence of apoptotic cells (8.3 ± 1.1 vs 5.3 ± 1.7, P < 0.01) were also increased significantly in rats with EGDA and concomitant esophageal H. pylori colonization compared with rats with EGDA only. The mRNA levels of cyclin D1 (5.8 ± 1.9 vs 3.4 ± 1.3, P < 0.01), c-Myc (6.4 ± 1.7 vs 3.7 ± 1.2, P < 0.01), and Bax (8.6 ± 1.6 vs 5.1 ± 1.3, P < 0.01) were significantly increased, whereas the mRNA level of Bcl-2 (0.6 ± 0.3 vs 0.8 ± 0.3, P < 0.01) was significantly reduced in rats with EGDA and concomitant esophageal H. pylori colonization compared with rats with EGDA only.CONCLUSION: Esophageal H. pylori colonization increases esophagitis severity, and facilitates the development of BE and EAC with the augmentation of cell proliferation and apoptosis in esophageal mucosa.     

Inhibitory effects of rapamycin on the different stages of hepatic fibrosis

AIM: To investigate and compare the inhibitory effects of rapamycin in the different stages of liver fibrosis.METHODS: We performed bile duct ligation (BDL) in male Wistar rats (n = 24). The experimental rats were classified into four groups: the BDL⁺/Rapa^- group (un-treated control, n = 4), the BDL⁺/Rapa⁺ group (treated 14 d after BDL, n = 8), the BDL⁺/Rapa⁺⁺ group (treated on the day after BDL, n = 8), and the BDL^-/Rapa^- group (un-treated, sham -operated control, n = 4). The BDL⁺/Rapa⁺ and BDL⁺/Rapa⁺⁺ groups were administered rapamycin (2 mg/kg) for 28 d. The liver tissues were tested by immunohistochemical staining for α-smooth muscle actin (α-SMA) and cytokeratin.RESULTS: The liver mRNA levels of transforming growth factor (TGF)-β1 and platelet-derived growth factor (PDGF) were measured using the polymerase chain reaction. The protein levels of liver p70s6K and p-p70s6k were determined using Western blotting. α-SMA expression was lowest in the BDL⁺/Rapa⁺⁺group. TGF-β1 and PDGF expression levels in the rapamycin-treated group were lower than those in the un-treated group and higher than those in the control groups (TGF-β1: 0.23 ± 0.00 vs 0.34 ± 0.01, 0.23 ± 0.0 vs 0.09 ± 0.00, P < 0.0001; PDGF: 0.21 ± 0.00 vs 0.34 ± 0.01, 0.21 ± 0.0 vs 0.09 ± 0.00, P < 0.0001). The p70s6k and p-p70s6k levels decreased in the treated groups and were lowest in the BDL⁺/Rapa⁺⁺group (p70s6k: 1.05 ± 0.17 vs 1.30 ± 0.56, 0.40 ± 0.01 vs 1.30 ± 0.56, P < 0.0001; p-p70s6k: 1.40 ± 0.5 vs 1.67 ± 0.12, 0.70 ± 0.01 vs 1.67 ± 0.12, P < 0.0001).CONCLUSION: The results of our study indicate that rapamycin has inhibitory effects on liver fibrosis, and the treatment is most effective in the early stages of fibrosis.     

High expression of CD11c indicates favorable prognosis in patients with gastric cancer

AIM: To determine the relationship between CD11 c expression level and prognosis in patients with gastric cancer(GC).METHODS: This retrospective survival study was performed from July 31,2008 to June 30,2014. Our study inclusion criteria included all the patients with GC who underwent surgical resection between January 1998 and December 2009 in the Third Affiliated Hospital of Soochow University. CD11 c expression levels in 140 patients with GC at different UICC stages were evaluated using immunohistochemistry,and GC tissues from 16 cases were further verified by q RTPCR. The χ2 test was used to compare the patientand disease-related factors between the low CD11 c expression group and the high expression group. Univariate probabilities of overall survival(OS) and disease-free survival(DFS) were assessed using the Kaplan-Meier method. The log rank test was used to compare survival curves. Different multivariate COX models were used to estimate the association between CD11 c expression and both death and recurrence riskin GC patients.RESULTS: The average CD11 c expression level was 5.1 ± 1.8/high power field(HPF) in 10 gastritis samples,4.5 ± 2.3/HPF in 10 gastric polyp samples and 9.7 ± 6.3/HPF in 140 gastric cancer samples,respectively. The CD11 c expression level was significantly decreased from UICC stage Ⅰ to stage Ⅳ(stage Ⅰ: 16.0 ± 7.4,stage Ⅱ: 10.4 ± 5.5,stage Ⅲ: 9.4 ± 6.1,stage Ⅳ: 5.3 ± 3.2,P 0.001). Patients in the high CD11 c expression group had a greater 3- and 5-year OS probability and longer median survival time compared with the low CD11 c expression group,(67.7% vs 39.2%; 51.4% vs 29.0%; 67.0 mo vs 28.0 mo; χ2 = 6.80,P = 0.009),and had a greater 3- and 5-year DFS probability and longer median DFS time(63.7% vs 24.0%; 49.1% vs 11.9%; 64.0 mo vs 18.0 mo; χ2 = 15.39,P 0.001). Patients with high CD11 c high expression had a reduced risk of death(HR = 0.56,95%CI: 0.33-0.98,P 0.05) and relapse(HR = 0.39,95%CI: 0.23-0.67,P 0.01) compared with patients with low CD11 c expression after adjustment of potential confounders,with the exception of tumor size. However,the protective effect related to death(HR = 0.90,95%CI: 0.49-1.67,P = 0.749) and relapse(HR = 0.65,95%CI: 0.36-1.19,P = 0.160) disappeared when tumor size was incorporated into the model.CONCLUSION: High expression of CD11 c decreased the risk of death and relapse,and may be regarded as an alternative indicator of favorable prognosis in patients with GC.     

Activated rat hepatic stellate cells influence Th1/Th2 profile in vitro

AIM: To investigate the effects of activated rat hepatic stellate cells(HSCs) on rat Th1/Th2 profile in vitro.METHODS: Growth and survival of activated HSCs and CD4+ T lymphocytes cultured alone or together was assessed after 24 or 48 h. CD4+ T lymphocytes were then cultured with or without activated HSCs for 24 or 48 h and the proportion of Th1 [interferon(IFN)-γ+] and Th2 [interleukin(IL)-4+] cells was assessed by flow cytometry. Th1 and Th2 cell apoptosis was assessed after 24 h of co-culture using a caspase-3 staining procedure. Differentiation rates of Th1 and Th2 cells from CD4+ T lymphocytes that were positive for CD25 but did not express IFN-γ or IL-4 were also assessed after 48 h of co-culture with activated HSCs. Galectin-9 expression in HSCs was determined by immunofluorescence and Western blotting. ELISA was performed to assess galectin-9 secretion from activated HSCs.RESULTS: Co-culture of CD4+ T lymphocytes with activated rat HSCs for 48 h significantly reduced the proportion of Th1 cells compared to culture-alone conditions(-1.73% ± 0.71%; P 0.05), whereas the proportion of Th2 cells was not altered; the Th1/Th2 ratio was significantly decreased(-0.44 ± 0.13; P 0.05). In addition, the level of IFN-γ in Th1 cells wasdecreased(-65.71 ± 9.67; P 0.01), whereas the level of IL-4 in Th2 cells was increased(82.79 ± 25.12; P 0.05) by co-culturing, as measured by mean fluorescence intensity by flow cytometry. Apoptosis rates in Th1(12.27% ± 0.99%; P 0.01) and Th2(1.71% ± 0.185%; P 0.01) cells were increased 24 h after co-culturing with activated HSCs; the Th1 cell apoptosis rate was significantly higher than in Th2 cells(P 0.01). Galectin-9 protein expression was significantly decreased in HSCs only 24 h after coculturing(P 0.05) but not after 48 h. Co-culture for 48 h significantly increased the differentiation of Th1 and Th2 cells; however, the increase in the proportion of Th2 cells was significantly higher than that of Th1 cells(1.85% ± 0.48%; P 0.05).CONCLUSION: Activated rat HSCs lower the Th1/Th2 profile, inhibiting the Th1 response and enhancing the Th2 response, and this may be a novel pathway for liver fibrogenesis.     

Aloe vera attenuated gastric injury on indomethacin-induced gastropathy in rats

AIM: To evaluate the protective effects of Aloe vera on gastric injury in rats with indomethacin (IMN)-induced gastropathy.METHODS: Male Sprague-Dawley rats were randomly divided into three groups. Group 1 (control, n = 6) was given distilled water (DW) orally. Group 2 (IMN, n = 6) was given oral IMN (150 mg/kg) dissolved in 5% sodium bicarbonate (NaHCO₃^-) at time 0 and 4 h. Group 3 (Aloe vera-treated, n = 6) was given oral Aloe vera (150 mg/kg) dissolved in DW and IMN at time 0 and 4 h. Eight hours later, the stomach was removed to determine gastric malondialdehyde (MDA), the number of interleukin (IL)-18 positive stained cells (%) by immunohistochemistry, and for histopathological examination. Then, the serum was collected to determine tumor necrosis factor (TNF)-α and cytokine-induced neutrophil chemoattractant (CINC)-1 by sandwich enzyme linked immunosorbent assay method.RESULTS: In the IMN group, serum TNF-α, CINC-1 and gastric MDA were significantly increased when compared to the control group (27.78 ± 1.52 pg/mL vs 85.07 ± 49.11 pg/mL, P = 0.009; 104.55 ± 45.80 pg/mL vs 1054.70 ± 20.38 pg/mL, and 1.74 ± 0.21 nmol/mg vs 9.36 ± 1.07 nmol/mg protein, P = 0.000, respectively). The mean level of TNF-α, CINC-1 and gastric MDA in the Aloe vera-treated group were improved as compared with the IMN group (85.07 ± 49.11 pg/mL vs 35.19 ± 1.61 pg/mL, P = 0.021; 1054.70 ± 20.38 pg/mL vs 813.56 ± 239.04 pg/mL, P = 0.025; and 9.36 ± 1.07 nmol/mg vs 2.67 ± 0.64 nmol/mg protein, P = 0.000, respectively). The number of IL-18 positive stained cells (%) in the gastric epithelial cells of the IMN group was significantly higher than the control group (5.01% ± 3.73% vs 30.67% ± 2.03%, P = 0.000, respectively). In contrast, Aloe vera treatment decreased the number of IL-18 positive stained cells (%) significantly when compared with the IMN group (30.67% ± 2.03% vs 13.21% ± 1.10%, P = 0.000, respectively). Most rats in the IMN group developed moderate to severe gastric inflammation and erosions. The gastric erosions and neutrophil infiltration scores were significantly reduced in the Aloe vera-treated group.CONCLUSION: Aloe vera attenuated IMN-induced gastropathy in rats by the reduction of oxidative stress, inflammation, and improvement of gastric histopathology.     

Viral infection parameters not nucleoside analogue itself correlates with host immunity in nucleoside analogue therapy for chronic hepatitis B

AIM: To determine the relationship between host immunity and the characteristics of viral infection or nucleoside analogues (NAs) themselves in patients with chronic hepatitis B (CHB) receiving NA therapy.METHODS: Fifty-two hepatitis B envelope antigen (HBeAg) positive CHB patients were enrolled and divided equally into two groups. One group received telbivudine (LDT, 600 mg/d), and the other group received lamivudine (LAM, 100 mg/d). Clinical, virological and immunological parameters were assessed at the baseline and at 4, 12, 24, 36 and 48 wk.RESULTS: Both groups achieved significant hepatitis B virus (HBV) replication inhibition and alanine aminotransferase normalization at 48 wk. At the baseline, compared to healthy controls, CHB patients had a lower circulating CD8 T cell frequency (29.44% ± 11.55% vs 37.17% ± 7.30%, P = 0.03) and higher frequencies of programmed death 1 positive CD8 T cells (PD-1+ CD8 T) (16.48% ± 10.82% vs 7.02% ± 3.62%, P = 0.0001) and CD4+ CD25+ FoxP3+ T regulatory cells (Tregs) (23.64% ± 9.38% vs 13.60% ± 6.06%, P = 0.001). On therapy, at the beginning 24 wk with the levels of hepatitis B virus deoxyribonucleic acid (HBV DNA) and HBeAg declining, the frequencies of PD-1+ CD8 T cells and Treg cells gradually and significantly declined at 12 and 24 wk in both therapy groups. At treatment week 4, patients treated with LDT had a lower frequency of PD-1+ CD8 T cells compared to patients treated with LAM (10.08% ± 6.83% vs 20.51% ± 20.96%, P = 0.02). The frequency of PD-1+ CD8 T cells in all of the CHB patients was significantly correlated with both the HBV DNA level (r = 0.45, P = 0.01) and HBeAg level (r = 0.47, P = 0.01) at treatment week 24, but the frequency of Treg cells was only significantly correlated with the HBeAg level (r = 0.44,P = 0.02). Furthermore, the ability of CD8 T cells to secrete pro-inflammatory cytokines was partially restored after 24 wk of therapy.CONCLUSION: NA-mediated HBV suppression could down-regulate the production of negative regulators of host immunity during the first 24 wk of therapy and could partially restore the ability of CD8 T cells to secrete pro-inflammatory cytokines. This immune modulating response may be correlated with the levels of both HBV DNA and HBeAg.     

Abundance and significance of neuroligin-1 and glutamate in Hirschsprung’s disease

AIM: To investigate the abundance and potential diagnostic significance of neuroligin-1 and glutamate(Glu) in Hirschsprung's disease(HSCR).METHODS: Ninety children with HSCR and 50 children without HSCR matched for similar nutritional status, age and basal metabolic index were studied. The expression and localization of neuroligin-1 and Glu were assessed using double-labeling immunofluorescence staining of longitudinal muscles with adherent myenteric plexus from the surgically excised colon of children with HSCR. Western blot analysis, quantitative real-time PCR(q RT-PCR) and immunohistochemistry were performed to evaluate the abundance of neuroligin-1 and Glu in different HSCR-affected segments(ganglionic, transitional, and aganglionic segments). Enzyme-linked immunosorbent assay(ELISA) was used to detect and compare serum Glu levels in the long-segment HSCR, short-segment HSCR and non-HSCR samples.RESULTS: Neuroligin-1 and Glu were co-expressed highest to lowest in the ganglionic, transi tional and aganglionic segments based on Western blot(neuroligin-1: 0.177 ± 0.008 vs 0.101 ± 0.006, 0.177 ± 0.008 vs 0.035 ± 0.005, and 0.101 ± 0.006 vs 0.035 ±0.005, P 0.005; Glu: 0.198 ± 0.006 vs 0.115 ± 0.008, 0.198 ± 0.006 vs 0.040 ± 0.003, and 0.115 ± 0.008 vs 0.040 ± 0.003, P 0.005) and q RT-PCR(neuroligin-1: 9.58 × 10-5 ± 9.94 × 10-6 vs 2.49 × 10-5 ± 1.38 × 10-6, 9.58 × 10-5 ± 9.94 × 10-6 vs 7.17 × 10-6 ± 1.12 × 10-6, and 2.49 × 10-5 ± 1.38 × 10-6 vs 7.17 × 10-6 ± 1.12 × 10-6, P 0.005). Serum Glu level was the highest to lowest in the non-HSCR, short-type HSCR and long-type HSCR samples based on ELISA(in nmol/μL, 0.93 ± 0.31 vs 0.57 ± 0.25, 0.93 ± 0.31 vs 0.23 ± 0.16, and 0.57 ± 0.25 vs 0.23 ± 0.16, P 0.005).CONCLUSION: Neuroligin-1 and Glu may represent new markers of ganglion cells, whose expression may correlate with the pathogenesis, diagnosis, differential diagnosis or classification of HSCR.     

Intravoxel incoherent motion diffusion-weighted imaging for monitoring chemotherapeutic efficacy in gastric cancer

AIM: To assess intravoxel incoherent motion diffusionweighted imaging(IVIM-DWI) for monitoring early efficacy of chemotherapy in a human gastric cancer mouse model.METHODS: IVIM-DWI was performed with 12 b-values(0-800 s/mm2) in 25 human gastric cancer-bearing nude mice at baseline(day 0), and then they were randomly divided into control and 1-, 3-, 5- and 7-d treatment groups(n = 5 per group). The control group underwent longitudinal MRI scans at days 1, 3, 5 and 7, and the treatment groups underwent subsequent MRI scans after a specified 5-fluorouracil/calciumfolinate treatment. Together with tumor volumes(TV), the apparent diffusion coefficient(ADC) and IVIM parameters [true water molecular diffusion coefficient(D), perfusion fraction(f) and pseudo-related diffusion coefficient(D*)] were measured. The differences in those parameters from baseline to each measurement(ΔTV%, ΔADC%, ΔD%, Δf% and ΔD*%) were calculated. After image acquisition, tumor necrosis, microvessel density(MVD) and cellular apoptosis were evaluated by hematoxylin-eosin(HE), CD31 and terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL) staining respectively, to confirm the imaging findings. Mann-Whitney test and Spearman's correlation coefficient analysis were performed.RESULTS: The observed relative volume increase(ΔTV%) in the treatment group were significantly smaller than those in the control group at day 5(ΔTV_(treatment)% = 19.63% ± 3.01% and ΔTVcontrol% = 83.60% ± 14.87%, P = 0.008) and day 7(ΔTV_(treatment)% = 29.07% ± 10.01% and ΔTV_(control)% = 177.06% ± 63.00%, P = 0.008). The difference in ΔTV% between the treatment and the control groups was not significant at days 1 and 3 after a short duration of treatment. Increases in ADC in the treatment group(ΔADC%_(treatment), median, 30.10% ± 18.32%, 36.11% ± 21.82%, 45.22% ± 24.36%) were significantly higher compared with the control group(ΔADC%_(control), median, 4.98% ± 3.39%, 6.26% ± 3.08%, 9.24% ± 6.33%) at days 3, 5 and 7(P = 0.008, P = 0.016, P = 0.008, respectively). Increases in D in the treatment group(ΔD%_(treatment), median 17.12% ± 8.20%, 24.16% ± 16.87%, 38.54% ± 19.36%) were higher than those in the control group(ΔD%_(control), median-0.13% ± 4.23%, 5.89% ± 4.56%, 5.54% ± 4.44%) at days 1, 3, and 5(P = 0.032, P = 0.008, P = 0.016, respectively). Relative changes in f were significantly lower in the treatment group compared with the control group at days 1, 3, 5 and 7 follow-up(median,-34.13% ± 16.61% vs 1.68% ± 3.40%, P = 0.016;-50.64% ± 6.82% vs 3.01% ± 6.50%, P = 0.008;-49.93% ± 6.05% vs 0.97% ± 4.38%, P = 0.008, and-46.22% ± 7.75% vs 8.14% ± 6.75%, P = 0.008, respectively). D* in the treatment group decreased significantly compared to those in the control group at all time points(median,-32.10% ± 12.22% vs 1.85% ± 5.54%, P = 0.008;-44.14% ± 14.83% vs 2.29% ± 10.38%, P = 0.008;-59.06% ± 19.10% vs 3.86% ± 5.10%, P = 0.008 and-47.20% ± 20.48% vs 7.13% ± 9.88%, P = 0.016, respectively). Furthermore, histopathologic findings showed positive correlations with ADC and D and tumor necrosis(r_s = 0.720, P 0.001; r_s = 0.522, P = 0.007, respectively). The cellular apoptosis of the tumor also showed positive correlations with ADC and D(r_s = 0.626, P = 0.001; r_s = 0.542, P = 0.005, respectively). Perfusionrelated parameters(f and D*) were positively correlated to MVD(r_s = 0.618, P = 0.001; r_s = 0.538, P = 0.006, respectively), and negatively correlated to cellular apoptosis of the tumor(r_s =-0.550, P = 0.004; r_s =-0.692, P 0.001, respectively).CONCLUSION: IVIM-DWI is potentially useful for predicting the early efficacy of chemotherapy in a human gastric cancer mouse model.     

Comparison of mammosphere formation from breast cancer cell lines and primary breast tumors

Background

Breast cancer stem cells (BCSCs) can be enriched by culturing of cells in non-adherent non-differentiating conditions. However, culturing mammospheres from primary breast tumors are costly and difficult to control. In order to overcome problems associated with using primary human tissues, continuous breast cancer cell lines have been developed from various sources.

Methods

In this study, a luminal subtype breast cancer cell line MCF-7 and a basal subtype cell line MDA-MB-231 were chosen. We explored the optimal culturing system for BCSCs from the two cell lines and primary breast tumors. Then, mammosphere formation efficiency (MFE), CD44⁺/CD24^–/lowESA⁺Lin^– cell proportion in mammospheres, and tumorigenecity of mammospheres generated from the two breast cancer cell lines and primary breast tumors were compared.

Results

Enzymatic digestion of 60 mins and the addition of B27 to the culture medium were optimal for mammosphere culturing. Mammospheres could be formed in all the three cells, in which MCF-7 had the highest MFE. After 3 weeks culture, CD44⁺/CD24^–/lowESA⁺Lin^– cell proportion in mammospheres from MCF-7, MDA-MB-231 cells and primary breast tumors was 95.0%±2.5%, 82%±22% and 21.5%±1.0%, respectively. A total of 1,000 cells from MCF-7, MDA-MB-231 mammospheres but not primary mammospheres were tumorigenic.

Conclusions

This study validates the use of breast cancer cell lines as models to elucidate the nature of BCSCs.